This invention relates to a method of separating acids and/or proteins from a crude biological mixture. More particularly, this invention, in one of its embodiments, relates to a method of simultaneously separating nucleic acids and proteins from a crude biological mixture through the use of an aqueous salt solution of cesium trifluoroacetate.
In the fields of biochemistry, genetic engineering, molecular biology, medical research, and the like, it is often necessary to prepare pure samples of DNA and RNA from crude biological mixtures. (As used herein, the term "biological mixture" refers to a mixture of biological molecules, such as nucleic acids and proteins.) It is also important for workers in these fields to be able to isolate the DNA, RNA and protein fractions from crude biological mixtures. (As used herein, the term "protein" includes proteins as well as other polypeptides which may not be proteins.) Such crude biological mixtures are obtained when cells are lysed. The crude biological mixtures often contain low molecular weight compounds and carbohydrates as well as nucleic acids and proteins.
Heretofore, the technique known as "equilibrium density gradient centrifugation" has been used in the laboratory to isolate nucleic acids. In accordance with this technique, a crude or semi-purified biological mixture containing nucleic acids, proteins and other cellular components, is added to a dense salt solution and the resulting mixture is ultracentrifuged at very high gravity forces (e.g., 30,000 to 100,000 rpm) for periods of 1-3 days. Under the high centrifugal forces, the heavy salt ions are preferentially displaced outward, i.e., toward the bottom of the centrifuge vessel. A density gradient is thereby formed in the vessel with the liquid at the top of the vessel having a somewhat lower density than the liquid
bottom of the vessel. Ideally, each of the biological molecules in the vessel will seek its own buoyant density and band in a different location. For example, DNA will normally band at a density in the neighborhood of about 1.5 g/ml.
The most commonly used salts for DNA purification by this technique are cesium chloride (CsCl) and cesium sulfate (Cs.sub.2 SO.sub.4). These salts are sufficiently soluble in water to form solutions having a density of about 1.4-1.6 g/ml. Thus, these salts are adequate for isolating DNA from a crude biological mixture since DNA bands at about 1.5 g/ml. However, cesium chloride and cesium sulfate are inadequate for banding the RNA and protein fractions from a crude biological mixture. Due to the intense "salting-out" properties of the chloride and sulfate anions, proteins tend to precipitate from cesium chloride and cesium sulfate solutions and cannot be banded. In addition, the proteins which precipitate from solution cause some of the DNA to co-precipitate with them from solution. Therefore, crude mixtures must be thoroughly deproteinized before subjecting them to equilibrium density gradient ultracentrifugation in cesium chloride or cesium sulfate solutions.
Furthermore, RNA precipitates from and will not band in cesium chloride solution. While RNA will band in cesium sulfate solutions, RNA will precipitate out as well. Thus, neither cesium chloride nor cesium sulfate are satisfactory for simultaneously isolating DNA, RNA and protein fractions from crude or semi-purified biological mixtures.
Until recently, it has been necessary, in order to prepare pure samples of DNA by this technique, to free the DNA of protein by multiple extractions of the crude mixture with phenol or chloroform. It has also been necessary, in many cases, to treat the crude mixture with proteases in order to remove the proteins tightly bound to the DNA. It has also been necessary, in some cases, to treat the crude mixture with ribonuclease in order to degrade the RNA prior to its removal. These multiple treatments are time-consuming and inconvenient. The use of phenol is unpleasant and hazardous. The use of proteases and ribonuclease is fraught with difficulties because these enzymes can be easily contaminated with low levels of other enzymes.
Recently, aqueous solutions of cesium trichloroacetate ("CsTCA") have been proposed for the isolation of nucleic acids and proteins by the technique of equilibrium density gradient centrifugation. See Burke, R.L., and Bauer, W.R., 5 Nucleic Acids Research, 4819 (1978), and Burke, R.L., Anderson, P.J., and Bauer, W.R. 86 Anal. Biochem. 264 (1978). These publications disclose that various DNAs and RNAs can be banded in neutral CsTCA solutions without formation of any precipitate. However, cesium trichloroacetate solutions have not proved ideal for isolating nucleic acids and proteins because of three major limitations: (1) CsTCA solutions absorb strongly in the same region of the ultraviolet spectrum where nucleic acids also absorb strongly, thus hindering conventional techniques for detecting the presence of nucleic acids; (2) CsTCA slowly hydrolyzes in the presence of water to form chloroform and hydrochloric acid, thus hindering its use in solutions due to pH fluctuations and preventing its storage as a solution; and (3) upon occasion, dry CsTCA has been known to explode unexpectedly.
Thus, there remains a need for a method for isolating substantially pure fractions of DNA, RNA and/or proteins from a crude or semi-purified biological mixture without suffering the limitations presented by cesium trichloroacetate.